| Project/Area Number |
17613001
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
極限環境生物学
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| Research Institution | Kobe University |
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Principal Investigator |
TSURUTA Hiroki Kobe University, Headquarter of Innovative Cooperation and Development, Associate Professor, 連携創造本部, 助教授 (20346282)
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| Co-Investigator(Kenkyū-buntansha) |
MIKAMI Bunzo Kyoto University, Graduate School of Agriculture, Professor, 農学研究科, 教授 (40135611)
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| Project Period (FY) |
2005 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2005: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | low temperature environment / cold-active enzyme / phosphatase / X-ray structural analysis / hydrophobic interaction / 低温活性機構 / 変異型酵素解析 / 高分解能X線結晶構造解析 / 変異体ライブラリー |
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| Research Abstract |
Our final goal is that the investigation of the structural factor(s) for eliciting the highly catalytic efficiency of cold-active enzymes. For the purpose, we focused on cold-active protein-tyorosine phopshtase (CAPTPase) of a psychrophile Shewanella sp. as a experimental material. CAPTPase is a novel protein tyrosine phosphatase with the phosphoesterase motif conserved among metallophosphoesterases including many protein serine/threonine phosphatases. In this study, firstly, in order to reveal the detailed structural features and the reaction mechanism of this enzyme, we specified the metal ions in the enzyme protein and determined the crystal structure of CAPTPase at resolution of 1.1 Å. As the results, it was obvious that CAPTPase had two Zn atoms (termed Zn1 and Zn2) constructing a dinuclear Zn-Zn center in the catalytic site and that the hydrolysis of this enzyme was initiated by the nucleophilic attack with a water molecule bridging two Zn atoms after the substrate binding. The loop structures in the dinuclear Zn-Zn center were lined mainly with hydrophobic interactions. The mutational analysis for these hydrophobic residues and the determined crystal structure of I115M mutant enzyme, of which the catalytic efficiency was higher than that of the wild type enzyme, indicated that the catalysis of CAPTPase was dependent on the chemical bond force of hydrophobic interactions behind the loop structures contributing to the configuration of Zn1 atom.
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